TaqManast Advanced Master Mix F - Thermo Fisher Scientific€¦ · TaqManast Advanced Master Mix...

For Research Use Only. Not for use in diagnostic procedures.

TaqMan® Fast Advanced Master MixUSER GUIDE

For two-step RT-PCR in gene expression experiments orquantitative analysis

Catalog Numbers 4444556, 4444557, 4444558, 4444963, 4444964, 4444965Publication Number 4444605

Revision D

Manufacturer: Life Technologies Corporation | 2130 Woodward Street | Austin, TX 78744

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL,INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOURUSE OF IT.

Revision history: Pub. No. 4444605

Revision Date DescriptionD 20 July 2017 Remove the UNG incubation for standard microRNA assays and advanced

miRNA assays; change the PCR Enzyme Activation step to 2 minutes forGene Expression Assays (single tube and plate).

C 21 March 2017 Update licensing, trademarks, general style and format; updateinstrumentation; separate miRNA assay chapters.

B June 2010 Baseline for this revision history.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you acceptthe terms and conditions of all applicable Limited Use Label Licenses.Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registeredtrademark of Roche Molecular Systems, Inc., used under permission and license. Eppendorf and LoBind are trademarks of Eppendorf AG. Pipetmanis a trademark of Gilson S.A.S.

©2017 Thermo Fisher Scientific Inc. All rights reserved.

Contents

About this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Purpose of this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

■ CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

■ CHAPTER 2 RT-PCR for TaqMan® and Custom TaqMan® GeneExpression Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Perform reverse transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Prepare the PCR reaction mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Prepare the PCR reaction plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Set up a plate document or experiment file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Run the PCR reaction plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Analyze data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

■ CHAPTER 3 RT-PCR for TaqMan® Gene Expression Array Plates . . 16



Analyze data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

TaqMan® Fast Advanced Master Mix User Guide 3

■ CHAPTER 4 RT-PCR for TaqMan® Gene Expression Array Cards . . . 19


Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Recommended amount of cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Prepare the sample-specific PCR reaction mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Prepare the TaqMan® Array Card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Set up a card document or experiment file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Run the TaqMan® Array Card . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Analyze data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

■ CHAPTER 5 RT-PCR for TaqMan® MicroRNA Assays . . . . . . . . . . . . . . . . 23



Analyze data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

■ CHAPTER 6 RT-PCR for TaqMan® Advanced miRNA Assays . . . . . . . . 27


Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Prepare PCR reaction mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Prepare the PCR reaction plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Set up plate document or experiment file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29Run the PCR reaction plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Analyze data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Contents

4 TaqMan® Fast Advanced Master Mix User Guide

■ APPENDIX A Background information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Components of the TaqMan® Fast Advanced Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31AmpliTaq™ Fast DNA Polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Uracil-N glycosylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31dUTP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31ROX™ Passive Reference dye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Two-step RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

About the 5' nuclease assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33TaqMan® MGB probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

■ APPENDIX B Best practices for PCR and RT-PCR experiments . . . . 35

Good laboratory practices for PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Use UNG to prevent false-positive amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Detect fluorescent contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

■ APPENDIX C Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

■ Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Contents


About this guide

IMPORTANT! Before using this product, read and understand the information in the“Safety” appendix in this document.

Purpose of this guide

The TaqMan® Fast Advanced Master Mix User Guide describes how to perform two-stepRT-PCR using TaqMan® Fast Advanced Master Mix with:

• TaqMan® Gene Expression Assays, Custom TaqMan® Gene Expression Assays,and custom TaqMan® primer and probe sets

• TaqMan® Gene Expression Array Plates (Fast and Standard plates)• TaqMan® Gene Expression Array Cards• TaqMan® MicroRNA Assays• TaqMan® Advanced miRNA Assays

This User Guide provides general guidelines for analyzing data because analysis canvary between applications. See the documentation for your instrument for moreinformation about procedures and data analysis.


Product information

Product description

The Applied Biosystems™ TaqMan® Fast Advanced Master Mix enables PCR in anygene expression experiment or quantitative analysis, including:

• Pathogen detection• Differential gene expression analysis• Viral load quantitation• MicroRNA quantitation• Microarray verification

TaqMan® Fast Advanced Master Mix can be used with any DNA target, includingcomplementary DNA (cDNA) or genomic DNA (gDNA). It can be used in the secondstep of a two-step RT-PCR protocol for RNA quantitation experiments. A cDNAtemplate can be generated from RNA using one of our reverse transcription kits (see “Required materials not supplied“ on page 8) prior to PCR with the TaqMan® FastAdvanced Master Mix.

TaqMan® Fast Advanced Master Mix is supplied at a 2X concentration and contains:• AmpliTaq™ Fast DNA Polymerase• Uracil-N glycosylase (UNG)• dNTPs with dUTP• ROX™ dye (passive reference)• Optimized buffer components

See “Components of the TaqMan® Fast Advanced Master Mix“ on page 31 for moreinformation about each component.

TaqMan® Fast Advanced Master Mix is optimized for use with primers and TaqMan®

probes designed according to our guidelines.

1


Contents and storage

Table 1 TaqMan® Fast Advanced Master Mix

Cat. No. Number of 20-µL reactions Amount Storage[1]

4444556 100 1 × 1 mL

–20°C; 4°C after opening

4444557 500 1 × 5 mL

4444963 (2 × 4444557) 1,000 2 × 5 mL

4444964 (5 × 4444557) 2,500 5 × 5 mL

4444965 (10 × 4444557) 5,000 10 × 5 mL

4444558 5,000 1 × 50 mL

[1] See label for expiration date.

Required materials not supplied

Unless otherwise indicated, all materials are available through thermofisher.com.MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.

Table 2 Instrument, software, equipment, plates and accessories, and consumables

Item Source

Instrument, one of the following:

QuantStudio™ 3 and 5 Real-Time PCR Instruments[1]

Contact your local sales office.

QuantStudio™ 6 Flex Real-Time PCR System[1]

QuantStudio™ 7 Flex Real-Time PCR System

QuantStudio™ 12K Flex Real-Time PCR System

StepOne™ Real-Time PCR System[2]

StepOnePlus™ Real-Time PCR System[1]

7500 Real-Time PCR System[1]

7500 Fast Real-Time PCR System[1]

ViiA™ 7 Real-Time PCR System

7900HT Real-Time PCR Instrument[1]

7900HT Fast Real-Time PCR Instrument

Or use a compatible real-time PCR instrument from another supplier.Validate thermal cycling conditions on other real-time PCR instruments.

Chapter 1 Product informationContents and storage1


http://www.thermofisher.com

http://fisherscientific.com

Item Source

Software

Microsoft™ Excel™ (Optional, to create plate layout files for import) microsoft.com

Equipment

Centrifuge with plate adapter MLS

Microcentrifuge MLS

Thermal cycler, or heat block or water bath set to 95°C MLS

Adjustable pipettors MLS

Laboratory mixer (vortex or equivalent) MLS

Tubes, plates, and other consumables

Plastics consumables thermofisher.com/plastics

Pipette tips thermofisher.com/pipettetips

Disposable gloves MLS

[1] Not compatible with TaqMan® Array Cards.[2] Not compatible with TaqMan® Array Plates or TaqMan® Array Cards.

Table 3 Reagents for reverse transcription

Item Source

Reagents for reverse transcription (all assays)

TE, pH 8.0 AM9849

(Optional) RNase InhibitorN8080119

AM2684 (Cloned; 40 U/µL)

Nuclease-Free Water (not DEPC-Treated) AM9930

Reagents for reverse transcription (TaqMan® and Custom TaqMan® Gene Expression Assays, TaqMan® ArrayPlates, and TaqMan® Array Cards)

High-Capacity cDNA Reverse Transcription Kit4368814

4374966 (with RNase Inhibitor)

High-Capacity RNA-to-cDNA™ Kit 4387406

SuperScript™ VILO™ cDNA Synthesis Kit 11754050

SuperScript™ IV VILO™ Master Mix 11756050

Reagents for reverse transcription (TaqMan® MicroRNA Assays)

TaqMan® MicroRNA Reverse Transcription Kit[1] 4366596

Chapter 1 Product informationRequired materials not supplied 1


http://www.microsoft.com

http://www.thermofisher.com/plastics

http://www.thermofisher.com/pipettetips

Item Source

TaqMan® Advanced miRNA cDNA Synthesis Kit[2] A28007

[1] TaqMan® MicroRNA Assays are optimized for use with the TaqMan® MicroRNA Reverse Transcription Kit. Assay performance cannot be guaranteed with other reverse transcription kits.

[2] TaqMan® Advanced miRNA Assays are optimized for use with the TaqMan® Advanced miRNA cDNA Synthesis Kit. Assay performance cannot be guaranteed with other reverse transcription kits.

Table 4 Assays

Item Source

TaqMan® Assays

TaqMan® Gene Expression Assays thermofisher.com/taqmangeneexpression

Custom TaqMan® Gene Expression Assays thermofisher.com/taqmancustomgeneexpression

Custom TaqMan® probes and primers[1] thermofisher.com/customprimersprobes

TaqMan® Array Plates

TaqMan® 96-Well Array Plates (Standard and Fast) thermofisher.com/taqmanarrays

TaqMan® Array Cards

TaqMan® Array Card thermofisher.com/taqmanarrays

TaqMan® MicroRNA Assays

TaqMan® MicroRNA Assays thermofisher.com/taqmanmirna

TaqMan® Advanced miRNA Assays thermofisher.com/advancedmirna

Custom TaqMan® Small RNA Assays thermofisher.com/taqmancustommirna

[1] Synthesized to your sequence and choice of quencher and reporter dyes.

Table 5 Kits and reagents for RNA isolation

Item Source

RNA isolation products thermofisher.com/rnaisolation

Supporting reagents thermofisher.com/rnaisolationreagents

Chapter 1 Product informationRequired materials not supplied1


http://www.thermofisher.com/taqmangeneexpression

http://www.thermofisher.com/taqmancustomgeneexpression

http://www.thermofisher.com/customprimersprobes

http://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/taqman-gene-expression/real-time-pcr-taqman-arrays.html?CID=fl-WE112784

http://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/taqman-gene-expression/real-time-pcr-taqman-arrays.html?CID=fl-WE112784

http://www.thermofisher.com/taqmanmirna

http://www.thermofisher.com/advancedmirna

https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/mirna-ncrna-taqman-assays/custom-taqman-small-rna-assays.html

http://thermofisher.com/rnaisolation

http://thermofisher.com/rnaisolationreagents

Workflow

Perform reverse transcription

▼

Perform real-time PCR amplification

Prepare the PCR reaction mix

▼

Prepare the PCR reaction plate or TaqMan® Array Card

▼

Set up a plate or card document, or experiment file

(or use the document provided with the cards or custom plates)

▼

Run the PCR reaction plate or TaqMan® Array Card

▼

Analyze the data

Chapter 1 Product informationWorkflow 1


RT-PCR for TaqMan® and CustomTaqMan® Gene Expression Assays


Perform reverse transcription to obtain cDNA from RNA samples.See Table 3 for reverse transcription kits. See TaqMan® Gene Expression Assays UserGuide (Single-tube Assays) (Pub. No. 4333458) for detailed guidelines and instructions.

Perform PCR

• Store TaqMan® Assays frozen and away from light until use. Excessive exposureto light may affect the fluorescent probes.

• Multiple assays can be run on one reaction plate. Include no-template controls(NTCs) for each assay.

• Determine the total number of PCR reactions, including a gene expression assayfor each cDNA sample and a no-template control for each assay.

Note: We recommend three replicates reactions for each assay.

• Thaw the TaqMan® Fast Advanced Master Mix on ice, then mix thoroughly butgently.

• Thaw TaqMan® Assays on ice, then vortex and briefly centrifuge to resuspend.

• Thaw samples on ice, then vortex and briefly centrifuge to resuspend.

2

Guidelines

Before you begin


1. Combine the following components for the number of reactions required, plus10% overage.

ComponentVolume per reaction

Finalconcentration384-well plate 96- or 48-well

plates[1]

TaqMan® Fast AdvancedMaster Mix (2X)

5.0 µL 10.0 µL 1X

TaqMan® Assay (20X) 0.5 µL 1.0 µL 1X

Nuclease-Free Water[2] 3.5 µL 7.0 µL —

Total volume per reaction 9.0 µL 18.0 µL —

[1] Standard and Fast.[2] Adjust the volume of Nuclease-Free Water for a larger volume of cDNA.

2. Vortex briefly to mix.

3. Centrifuge briefly to bring the reaction mix to the bottom of the tube andeliminate air bubbles.

1. Transfer the appropriate volume of PCR reaction mix to each well of an opticalreaction plate.

2. Add cDNA template (1 pg to 100 ng in Nuclease-Free Water), or Nuclease-FreeWater for NTC, to each well.

• 1.0 µL for a 384-well plate• 2.0 µL for 96- and 48-well plates (Standard and Fast)

Note: Be sure to adjust the volume of Nuclease-Free Water in the PCR reactionmix for a larger volume of cDNA.

3. Seal the reaction plate with optical adhesive film, then centrifuge briefly to bringthe PCR reaction mix to the bottom of the well and eliminate air bubbles.

4. Apply a compression pad to the plate, if required by your real-time PCR system.

Prepare the PCRreaction mix

Prepare the PCRreaction plate

Chapter 2 RT-PCR for TaqMan® and Custom TaqMan® Gene Expression AssaysPerform PCR 2


1. Set up a plate document or experiment file using the following conditions:

Real-time PCR system

UNG incubation[1] Polymeraseactivation[2] PCR (40 cycles)

Hold50°C

Hold95°C

Denature95°C

Anneal / extend60°C

• QuantStudio™ 3 and 5 Real-TimePCR Instruments

• QuantStudio™ 6 and 7 Flex Real-Time PCR System

• QuantStudio™ 12K Flex Real-Time PCR System

• 7900HT Real-Time PCRInstrument

• 7900HT Fast Real-Time PCRInstrument

• ViiA™ 7 Real-Time PCR System

• StepOne™ Real-Time PCRSystem

• StepOnePlus™ Real-Time PCRSystem

2 minutes 2 minutes 1 second 20 seconds

• 7500 Fast Real-Time PCRSystem

• 7500 Real-Time PCR System

2 minutes 2 minutes 3 seconds 30 seconds

[1] For optimal UNG activity.[2] To activate AmpliTaq™ Fast DNA Polymerase.

2. Select the appropriate block, if this option applies to your instrument.

3. Select the appropriate experiment type, if this option applies to your instrument.

4. Select TaqMan® Reagents to detect the target sequence, if this option applies toyour instrument.

Set up a platedocument orexperiment file

Chapter 2 RT-PCR for TaqMan® and Custom TaqMan® Gene Expression AssaysPerform PCR2


5. Select a run mode.

Real-time PCR system Run mode

• 7900HT Real-Time PCR Instrument

• 7900HT Fast Real-Time PCR Instrument (384-Well andStandard 96-Well Block Modules)


Standard

• QuantStudio™ 3 and 5 Real-Time PCR Instruments




• StepOne™ Real-Time PCR System

• StepOnePlus™ Real-Time PCR System

• 7900HT Fast Real-Time PCR Instrument (Fast 96-WellBlock Module)

• 7500 Fast Real-Time PCR System

Fast

6. Enter the sample volume, if this option applies to your instrument.• 384-well plate: 10.0 µL• 96- and 48-well plates (both Standard and Fast): 20.0 µL

1. Open the plate document or experiment file that corresponds to the reactionplate in the system software.

2. Load the reaction plate.

3. Start the run.

Analyze data

Data analysis varies depending on your real-time PCR system. See the instrumentUser Guide for more information.

1. View the amplification plots for the reactions.

2. Use auto baseline and auto threshold settings, or set the baseline and thresholdvalues to determine the threshold cycles (Ct) for the amplification curves.

3. Use the relative standard curve method or the comparative Ct method to analyzedata.

Run the PCRreaction plate

Chapter 2 RT-PCR for TaqMan® and Custom TaqMan® Gene Expression AssaysAnalyze data 2


RT-PCR for TaqMan® GeneExpression Array Plates


Perform reverse transcription to obtain cDNA from RNA samples.See Table 3 for reverse transcription kits. See TaqMan® Gene Expression Assays UserGuide (Single-tube Assays) (Pub. No. 4333458) for detailed guidelines and instructions.

Perform PCR

Store TaqMan® Array Plates away from light until use. Excessive exposure to lightmay affect the fluorescent probes.

• Determine the total number of PCR reactions.One reaction corresponds to one well in the TaqMan® Array Plate.





96-well plate (Fast) 96-well plate(Standard)

cDNA template + Nuclease-FreeWater[1]

5 µL 10 µL

TaqMan® Fast Advanced MasterMix (2X)

5 µL 10 µL

Total volume per reaction 10 µL 20 µL

[1] 5–50 ng of cDNA diluted in Nuclease-Free Water.



3

Guidelines

Before you begin



1. Transfer the appropriate volume of PCR reaction mix to each well of a TaqMan®

Array Plate.

2. Seal the plate with optical adhesive film, then centrifuge briefly to bring the PCRreaction mix to the bottom of the well and eliminate air bubbles.





Hold50°C

Hold95°C

Denature95°C


• QuantStudio™ 3 and 5 Real-TimePCR Instruments



• 7900HT Real-Time PCRInstrument

• 7900HT Fast Real-Time PCRInstrument


• StepOnePlus™ Real-Time PCRSystem

2 minutes 2 minutes 1 second 20 seconds

• 7500 Fast Real-Time PCRSystem


2 minutes 2 minutes 3 seconds 30 seconds

[1] For optimal UNG activity.[2] To activate AmpliTaq™ Fast DNA Polymerase.






Chapter 3 RT-PCR for TaqMan® Gene Expression Array PlatesPerform PCR 3







Standard









Fast

6. Enter the sample volume, if this option applies to your instrument.• 96-well Fast plate: 10.0 µL• 96-well Standard plate: 20.0 µL



3. Start the run.

Analyze data



2. Set the baseline and threshold values to determine the threshold cycles (Ct) forthe amplification curves, or select relative threshold under analysis settings toobtain (Crt) values (recommended for dried-down assays).



Chapter 3 RT-PCR for TaqMan® Gene Expression Array PlatesAnalyze data3


RT-PCR for TaqMan® GeneExpression Array Cards


Perform reverse transcription to obtain cDNA from RNA samples.See “Required materials not supplied“ on page 8 for reverse transcription kits. See theprotocol for your kit and the TaqMan® Gene Expression Array Cards User Guide (Pub.No. 4400263) for detailed guidelines and instructions.

Perform PCR

• Store the TaqMan® Array Card in its packaging until the packaging has reachedroom temperature and you are ready to fill it with sample-specific PCR mix.Prolonged exposure to indoor lighting can photo-degrade the fluorescent probescontained within the card. Do not expose the card to sunlight.

• Fill each fill reservoir with sample-specific PCR mix made from a single cDNAsample.

• Use 100 µL of sample-specific PCR mix to fill each fill reservoir. Volumes smallerthan 100 µL will result in insufficiently filled cards.

• Do not add the sample after centrifuging the cards. Centrifugation of the cardcauses the sample-specific PCR mix to resuspend the dried TaqMan® probes andprimers within the wells of the card. Addition of the sample after centrifugingdisrupts the resuspended assay positions.

• Schedule runs so that each card is run as soon as possible, to ensurereproducibility. After sealing, there is no measurable well-to-well contaminationfor up to 64 hours.

• We recommend 30–1000 ng (0.3–10 ng/µL) of cDNA (converted from total RNA)per fill reservoir.

• The amount of cDNA to use depends on the expression level of the target genesand the number of target copies per well that need to be detected. For example:

– Use 1000 ng (10 ng/µL) per fill reservoir to detect genes with low expression.Because the cDNA concentration is high, use high-quality cDNA withoutinhibitors.

– Use 100–200 ng per fill reservoir to detect genes with moderate expression.– Use 30–50 ng per fill reservoir to detect genes with moderate to high

expression.• Use the same amount of cDNA sample for all reactions.

4

Guidelines

Recommendedamount of cDNA


• Determine the number of fill reservoirs in the TaqMan® Array Card that will beused for each cDNA sample.




Component Volume per fill reservoir

cDNA template + Nuclease-Free Water[1] 50 µL

TaqMan® Fast Advanced Master Mix (2X) 50 µL

Total volume 100 µL

[1] See “Recommended amount of cDNA“ on page 19.



Fill the TaqMan® Array Card with sample-specific PCR reaction mix, then centrifugeand seal (see the TaqMan® Gene Expression Array Cards User Guide; Pub. No. 4400263).

Set up a card document or experiment file for the TaqMan® Array Card.

Note: Thermal cycling conditions depend on the instrument.

• For the 7900HT Fast Real-Time PCR Instrument:– Thermal cycling conditions:


Hold50°C

Hold92°C

Denature97°C


2 minutes 10 minutes[3] 1 second 20 seconds

[1] For optimal UNG activity.[2] To activate AmpliTaq™ Fast DNA Polymerase.[3] To completely dissolve the primers and probes on the TaqMan® Array Card.

– Run mode: Standard

Before you begin

Prepare thesample-specificPCR reaction mix

Prepare theTaqMan® ArrayCard

Set up a carddocument orexperiment file

Chapter 4 RT-PCR for TaqMan® Gene Expression Array CardsPerform PCR4


– Ramp rate: 100%– Sample volume: 1.0 µL

• For the ViiA™ 7 Real-Time PCR System, the QuantStudio™ 7 Flex Real-Time PCRSystem, or the QuantStudio™ 12K Flex Real-Time PCR System:

– Select the appropriate template file for your real-time PCR system andexperiment:

Experiment type Template file

ViiA™ 7 Real-Time PCR System

Comparative Ct ViiA7_TaqMan_Array_Comparative_Ct_Fast_Template.edt

Standard Curve ViiA7_TaqMan_Array_Std_Curve_Fast_Template.edt

QuantStudio™ 7 Flex Real-Time PCR System and QuantStudio™ 12K Flex Real-Time PCR System

Comparative Ct TaqMan_Array_Comparative_Ct_Fast_Template.edt

Standard Curve TaqMan_Array_Std_Curve_Fast_Template.edt

– Manually select each option:– Block: Array Card Block– Experiment type: Comparative Ct or Standard Curve– Reagents: TaqMan® Reagents– Properties: Fast– Thermal cycling conditions, including ramp rate: Select Run Method,

then enter as indicated:


Hold50°C

Hold92°C

Denature95°C


2 minutes 10 minutes[3] 1 second 20 seconds

1.75°C/second 1.75°C/second 1.75°C/second 1.83°C/second

[1] For optimal UNG activity.[2] To activate AmpliTaq™ Fast DNA Polymerase.[3] To completely dissolve the primers and probes on the TaqMan® Array Card.

IMPORTANT! Do not use the default Fast settings.

1. Open the card document or experiment file that corresponds to the TaqMan®

Array Card in the system software.

2. Load the TaqMan® Array Card.

3. Start the run.

Run the TaqMan®

Array Card

Chapter 4 RT-PCR for TaqMan® Gene Expression Array CardsPerform PCR 4


Analyze data



2. Set the baseline and threshold values to determine the threshold cycles (Ct) forthe amplification curves, or select relative threshold under analysis settings toobtain (Crt) values (recommended for dried-down assays).


Chapter 4 RT-PCR for TaqMan® Gene Expression Array CardsAnalyze data4


RT-PCR for TaqMan® MicroRNAAssays

This chapter covers use of TaqMan® MicroRNA Assays. For use of TaqMan®

Advanced miRNA Assays see Chapter 6, “RT-PCR for TaqMan® Advanced miRNAAssays“.


Perform reverse transcription to obtain cDNA from RNA samples.Use the TaqMan® MicroRNA Reverse Transcription Kit, and see TaqMan® Small RNAAssays Protocol (Pub. No. 4364031) for detailed guidelines and instructions.

Perform PCR

• Store the TaqMan® MicroRNA Assays at –20°C and away from light until use.Excessive exposure to light may affect the fluorescent probes.

• Prepare the PCR reaction mix before transferring it to the reaction plate forthermal cycling.

• Determine the total number of PCR reactions, including a microRNA assay foreach cDNA sample, endogenous control assays, and a no-template control (NTC)for each assay.

Note: We recommend three replicate reactions for each assay.


5

Guidelines

Before you begin




384-well plate 96- or 48-wellplates[1]


5.00 µL 10.00 µL

Nuclease-Free Water[2] 3.83 µL 7.67 µL

TaqMan® MicroRNA Assay (20X) 0.50 µL 1.00 µL

cDNA[3] 0.67 µL 1.33 µL

Total volume per reaction 10.00 µL 20.00 µL

[1] Standard and Fast.[2] Adjust the volume of Nuclease-Free Water for a larger volume of cDNA.[3] Minimum final dilution of RT reaction in PCR reaction is 1:15.

2. Mix gently, then centrifuge to bring the reaction mix to the bottom of the tube.






Chapter 5 RT-PCR for TaqMan® MicroRNA AssaysPerform PCR5




Polymeraseactivation[1] PCR (40 cycles)

Hold95°C

Denature95°C


• QuantStudio™ 3 and 5 Real-Time PCRInstruments

• QuantStudio™ 6 and 7 Flex Real-TimePCR System

• QuantStudio™ 12K Flex Real-Time PCRSystem


• 7900HT Fast Real-Time PCR Instrument




20 seconds 1 second 20 seconds



20 seconds 3 seconds 30 seconds

[1] To activate AmpliTaq™ Fast DNA Polymerase.





Chapter 5 RT-PCR for TaqMan® MicroRNA AssaysPerform PCR 5







Standard









Fast




3. Start the run.

Analyze data



2. Use auto baseline and auto threshold settings, or set the baseline and thresholdvalues to determine the threshold cycles (Ct) for the amplification curves.



Chapter 5 RT-PCR for TaqMan® MicroRNA AssaysAnalyze data5


RT-PCR for TaqMan® AdvancedmiRNA Assays

This chapter covers use of TaqMan® Advanced miRNA Assays. For use of TaqMan®

MicroRNA Assays see Chapter 5, “RT-PCR for TaqMan® MicroRNA Assays“.


Perform reverse transcription to obtain cDNA from RNA samples.Use the TaqMan® Advanced miRNA cDNA Synthesis Kit, and see TaqMan® AdvancedmiRNA Assays User Guide (Single-tube Assays) (Pub. No. 100027897) for detailedguidelines and instructions.

Perform PCR

• Store the TaqMan® Advanced miRNA Assays at –20°C and away from light untiluse. Excessive exposure to light may affect the fluorescent probes.

• Prepare the PCR reaction mix before transferring it to the reaction plate forthermal cycling.

• Determine the total number of PCR reactions, including a microRNA assay foreach cDNA sample, endogenous control assays, and a no-template control (NTC)for each assay.

Note: We recommend three replicate reactions for each assay.


6

Guidelines

Before you begin


1. Prepare 1:10 dilutions of the cDNA template.



384-well plate 96- or 48-well plates[1]


5.00 µL 10.00 µL

Nuclease-Free Water[2] 2.00 µL 4.00 µL

TaqMan® Advanced miRNAAssay (20X)

0.50 µL 1.00 µL

cDNA (1:10 dilution) 2.50 µL 5.00 µL

Total volume per reaction 10.00 µL 20.00 µL

[1] Standard and Fast.[2] Adjust the volume of Nuclease-Free Water for a larger volume of cDNA.

3. Mix gently, then centrifuge to bring the reaction mix to the bottom of the tube.




Prepare PCRreaction mix


Chapter 6 RT-PCR for TaqMan® Advanced miRNA AssaysPerform PCR6




Polymeraseactivation[1] PCR (40 cycles)

Hold95°C

Denature95°C


• QuantStudio™ 3 and 5 Real-Time PCRInstruments

• QuantStudio™ 6 and 7 Flex Real-TimePCR System

• QuantStudio™ 12K Flex Real-Time PCRSystem


• 7900HT Fast Real-Time PCR Instrument




20 seconds 1 second 20 seconds



20 seconds 3 seconds 30 seconds

[1] To activate AmpliTaq™ Fast DNA Polymerase.




Set up platedocument orexperiment file

Chapter 6 RT-PCR for TaqMan® Advanced miRNA AssaysPerform PCR 6







Standard









Fast




3. Start the run.

Analyze data



2. Use auto baseline setting and threshold setting of 0.1, or set the baseline andthreshold values to determine the threshold cycles (Ct) for the amplificationcurves.



Chapter 6 RT-PCR for TaqMan® Advanced miRNA AssaysAnalyze data6


Background information

Components of the TaqMan® Fast Advanced Master Mix

The AmpliTaq™ Fast DNA Polymerase enzyme is purified through a proprietaryprocess to reduce bacterial DNA introduced from the host organism. The purificationprocess ensures that non-specific, false-positive DNA products due to bacterial DNAcontamination are minimized during PCR.

When AmpliTaq™ Fast DNA Polymerase is added to the reaction mixture at roomtemperature, the inactive enzyme is not capable of primer extension. Any low-stringency mispriming events that may have occurred will not be enzymaticallyextended and subsequently amplified. A thermal incubation step is required foractivation to ensure that active enzyme is generated only at temperatures where theDNA is fully denatured.

Uracil-N glycosylase (UNG) treatment can prevent the reamplification of carryoverPCR products by removing any uracil incorporated into single- or double-strandedamplicons (Longo et al., 1990). UNG prevents reamplification of carryover PCRproducts in an assay if all previous PCR for that assay was performed using a dUTP-containing master mix. See “Use UNG to prevent false-positive amplification“ onpage 35 for more information about UNG.

This master mix includes dUTP to enable uracil-N-glycosylase (UNG) activity andmaintain optimal PCR results.

The ROX™ Passive Reference dye provides an internal reference to which the reporterdye signal can be normalized during data analysis. Normalization is necessary tocorrect for fluorescent fluctuations due to changes in concentration or volume.

A

AmpliTaq™ FastDNA Polymerase

Uracil-Nglycosylase

dUTP

ROX™ PassiveReference dye


Two-step RT-PCR

Visit thermofisher.com/qpcreducation for more information.

A target template is a DNA sequence, including cDNA, a gDNA, or a plasmidnucleotide sequence. An amplicon is a short segment of DNA.

Gene quantitation assays using TaqMan® Fast Advanced Master Mix and TaqMan®

Assays are performed in a two-step RT-PCR.

1. In the reverse transcription (RT) step, cDNA is reverse transcribed from RNA.2. In the PCR step, PCR products are quantitatively synthesized from cDNA

samples using the TaqMan® Fast Advanced Master Mix.

5' 3'

3'

5'

5'

cDNA

cDNA

PCR Step

Extension of primer on cDNA

Synthesis of second cDNA strand

PCR amplification of cDNA

3'

5'

5'

5'

5'

5'

Forward primer

Forward primer

Reverse primer

Cycle #1

Cycle #23'

3'

Extension of primer on mRNA

mRNA

RT Step

Synthesis of first cDNA strand

Random primer

3'

3'5'5'

5'

Figure 1 Two-step RT-PCR.This illustration does not show hybridization of the TaqMan® MGB probe. See “TaqMan® MGBprobes“ on page 34 for details on how the TaqMan® MGB probe is used in the PCR step.

Appendix A Background informationTwo-step RT-PCRA


http://www.thermofisher.com/qpcreducation

About the 5' nuclease assay

The 5' nuclease assay process takes place during PCR amplification. It occurs in everycycle and does not interfere with the exponential accumulation of product.

During the PCR, the TaqMan® MGB probe anneals specifically to a complementarysequence between the forward and reverse primer sites.

When the probe is intact (Figure 3 and Figure 4), the proximity of the reporter dye tothe quencher dye results in suppression of the reporter fluorescence primarily byFörster-type energy transfer (Förster, 1948; Lakowicz, 1983).

NFQ

MGB

R

P

= Nonfluorescent quencher

= Minor groove binder

= Reporter

= Hot-start DNA polymerase

Figure 2 Legend.

P

Forward primer

P

Reverse primer

R NFQ

MGBProbe

5'3'

3'5'

Figure 3 Polymerization.

NFQ MGBR

P

P

ForwardPrimer

TaqManMGB Probe

ReversePrimer

5'

5'

5'

5'

3'

3'3'

Figure 4 Strand displacement.

The DNA polymerase cleaves only probes that hybridize to the target (Figure 5).Cleavage separates the reporter dye from the quencher dye; the separation of thereporter dye from the quencher dye results in increased fluorescence by the reporter.The increase in fluorescence occurs only if the target sequence is complementary to

Appendix A Background informationAbout the 5' nuclease assay A


the probe and amplified during PCR. Because of these requirements, nonspecificamplification is not detected.

NFQ MGBR

P

ForwardPrimer

TaqManMGB Probe

ReversePrimer

5'

3'

5'

5'

5' 3'

3'

P

Figure 5 Cleavage.

Polymerization of the strand continues, but because the 3' end of the probe is blocked,no extension of the probe occurs during PCR (Figure 6).

NFQ MGBRForwardPrimer

TaqManMGB Probe

ReversePrimer

5'3'

5'

5'

5' 3'

3'

Figure 6 Completion of polymerization.

TaqMan® MGB probes contain:• A reporter dye (for example, FAM™) at the 5′ end of the probe

(Afonina et al., 1997; Kutyavin et al., 1997)• A non-fluorescent quencher (NFQ) dye at the 3′ end of the probe.

The NFQ dye does not fluoresce, which allows the real-time PCR system tomeasure the reporter dye contributions more accurately.

• A minor groove binder (MGB) at the 3´ end of the probe that:– Increases the melting temperature (Tm) without increasing the probe length.– Allows for the design of shorter probes.

TaqMan® MGBprobes

Appendix A Background informationAbout the 5' nuclease assayA


Best practices for PCR and RT-PCRexperiments

Good laboratory practices for PCR and RT-PCR

When preparing samples for PCR or RT-PCR amplification:• Wear clean gloves and a clean lab coat.

– Do not wear the same gloves and lab coat that you have previously usedwhen handling amplified products or preparing samples.

• Change gloves if you suspect that they are contaminated.• Maintain separate areas and dedicated equipment and supplies for:

– Sample preparation and reaction setup.– Amplification and analysis of products.

• Do not bring amplified products into the reaction setup area.• Open and close all sample tubes carefully. Avoid splashing or spraying samples.• Keep reactions and components capped as much as possible.• Use a positive-displacement pipettor or aerosol-resistant barrier pipette tips.• Clean lab benches and equipment periodically with 10% bleach solution or

DNAZap™ Solutions (Cat. No. AM9890).

Use UNG to prevent false-positive amplification

Carryover amplicons can result in false-positive amplification during PCR. Use amaster mix that contains uracil-N-glycosylase (UNG; also known as uracil-DNAglycosylase (UDG)) to degrade many contaminating carryover amplicons.

UNG enzymatic activity occurs during an initial incubation at 50°C. UNG is partiallyinactivated during the 95°C incubation step for template denaturation andpolymerase activation. Because UNG is not completely deactivated during the 95°Cincubation, it is important to keep the annealing temperatures greater than 55°C andto refrigerate PCR products at 2°C to 8°C in order to prevent amplicon degradation.

To ensure the desired UNG activity:• Use PCR components and thermal cycling conditions as specified.

UNG-containing master mixes incorporate the optimal concentration of UNG toprevent cross-contamination while not affecting real-time PCR performance.

• Do not attempt to use UNG-containing master mixes in subsequent amplificationof dU-containing PCR products, such as in nested-PCR protocols. The UNG willdegrade the dU-containing PCR products, preventing further amplification.

B


Although treatment with UNG can degrade or eliminate large numbers of carryoverPCR products, use good laboratory practices to minimize cross-contamination fromnon-dU-containing PCR products or other samples.

Detect fluorescent contaminants

Fluorescent contaminants can generate false positive results. To help detect thesecontaminants, we recommend including a No-Amplification Control reaction thatcontains sample, but no master mix.

After PCR, if the absolute fluorescence of the No-Amplification Control is greater thanthe fluorescence of the no template control (NTC), fluorescent contaminants may bepresent in the sample or in the heat block of the real-time PCR instrument.

Appendix B Best practices for PCR and RT-PCR experimentsDetect fluorescent contaminantsB


Safety

WARNING! GENERAL SAFETY. Using this product in a manner not specifiedin the user documentation may result in personal injury or damage to theinstrument or device. Ensure that anyone using this product has receivedinstructions in general safety practices for laboratories and the safetyinformation provided in this document.

· Before using an instrument or device, read and understand the safetyinformation provided in the user documentation provided by themanufacturer of the instrument or device.

· Before handling chemicals, read and understand all applicable Safety DataSheets (SDSs) and use appropriate personal protective equipment (gloves,gowns, eye protection, etc). To obtain SDSs, see the “Documentation andSupport” section in this document.

C


Chemical safety

WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards,ensure laboratory personnel read and practice the general safety guidelines forchemical usage, storage, and waste provided below. Consult the relevant SDSfor specific precautions and instructions:

· Read and understand the Safety Data Sheets (SDSs) provided by thechemical manufacturer before you store, handle, or work with any chemicalsor hazardous materials. To obtain SDSs, see the “Documentation andSupport” section in this document.

· Minimize contact with chemicals. Wear appropriate personal protectiveequipment when handling chemicals (for example, safety glasses, gloves, orprotective clothing).

· Minimize the inhalation of chemicals. Do not leave chemical containers open.Use only with adequate ventilation (for example, fume hood).

· Check regularly for chemical leaks or spills. If a leak or spill occurs, followthe manufacturer's cleanup procedures as recommended in the SDS.

· Handle chemical wastes in a fume hood.· Ensure use of primary and secondary waste containers. (A primary waste

container holds the immediate waste. A secondary container contains spillsor leaks from the primary container. Both containers must be compatiblewith the waste material and meet federal, state, and local requirements forcontainer storage.)

· After emptying a waste container, seal it with the cap provided.· Characterize (by analysis if necessary) the waste generated by the particular

applications, reagents, and substrates used in your laboratory.· Ensure that the waste is stored, transferred, transported, and disposed of

according to all local, state/provincial, and/or national regulations.· IMPORTANT! Radioactive or biohazardous materials may require special

handling, and disposal limitations may apply.

Appendix C SafetyChemical safetyC


Biological hazard safety

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids,infectious agents, and blood of humans and other animals have the potential totransmit infectious diseases. Conduct all work in properly equipped facilitieswith the appropriate safety equipment (for example, physical containmentdevices). Safety equipment can also include items for personal protection, suchas gloves, coats, gowns, shoe covers, boots, respirators, face shields, safetyglasses, or goggles. Individuals should be trained according to applicableregulatory and company/ institution requirements before working withpotentially biohazardous materials. Follow all applicable local, state/provincial,and/or national regulations. The following references provide generalguidelines when handling biological samples in laboratory environment.

· U.S. Department of Health and Human Services, Biosafety in Microbiologicaland Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC)21-1112, Revised December 2009; found at:www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf

· World Health Organization, Laboratory Biosafety Manual, 3rd Edition,WHO/CDS/CSR/LYO/2004.11; found at:www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Appendix C SafetyBiological hazard safety C


http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf

http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Documentation and support

Customer and technical support

Visit thermofisher.com/support for the latest in services and support, including:• Worldwide contact telephone numbers• Product support, including:

– Product FAQs– Software, patches, and updates– Training for many applications and instruments

• Order and web support• Product documentation, including:

– User guides, manuals, and protocols– Certificates of Analysis– Safety Data Sheets (SDSs; also known as MSDSs)

Note: For SDSs for reagents and chemicals from other manufacturers,contact the manufacturer.

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forthin the Life Technologies' General Terms and Conditions of Sale found on LifeTechnologies' website at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact LifeTechnologies at www.thermofisher.com/support.


http://thermofisher.com/support

http://www.thermofisher.com/us/en/home/global/terms-and-conditions.html

http://www.thermofisher.com/us/en/home/global/terms-and-conditions.html

http://www.thermofisher.com/support

References

Afonina I, Zivarts M, Kutyavin I, et al. (1997) Efficient priming of PCR with shortoligonucleotides conjugated to a minor groover binder. Nucleic Acid Res 25(13):2657–2660.

Delort AM, Duplaa AM, Molko D, et al. (1985) Excision of uracil residues in DNA:mechanism of action of Escherichia coli and Micrococcus luteus uracil-DNAglycosylases. Nucleic Acid Res 13(2):319–335.

Förster VT (1948) Zwischenmolekulare Energiewanderung und Fluoreszenz. AnnPhys (Leipzig) 2(1–2):55–75.

Higuchi RG and Ochman H (1989) Production of single-stranded DNA templates byexonuclease digestion following the polymerase chain reaction. Nucleic Acid Res17(14):5865.

Kutyavin IV, Lukhtanov EA, Gamper HB, et al. (1997) Oligonucleotides withconjugated dihydroyrroloindole tripeptides: base composition and backbone effectson hybridization. Nucleic Acid Res 25(18):3718–3723.

Kwok S and Higuchi R (1989) Avoiding false positives with PCR. Nature 339(6221):237–238.

Kwok S, Kellogg DE, McKinney N, et al. (1990) Effects of primer-template mismatcheson the polymerase chain reaction: human immunodeficiency virus type 1 modelstudies. Nucleic Acid Res 18(4):999–1005.

Lakowicz JR, editor (1983) Principles of Fluorescence Spectroscopy. New York (NY):Springer. p 496.

Longo MC, Berninger MS, Hartley JL (1990) Use of uracil DNA glycosylase to controlcarry-over contamination in polymerase chain reactions. Gene 93(1):125-128.

Mullis KB and Faloona FA (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:335–350.

Saiki RK, Gelfand DH, Stoffel S, et al. (1988) Primer-directed enzymatic amplificationof DNA with a thermostable DNA polymerase. Science 239(4839):487–491.

Saiki RK, Scharf S, Faloona F, et al. (1985) Enzymatic amplification of beta-globingenomic sequences and restriction site analysis for diagnosis of sickle cell anemia.Science 230(4732):1350–1354.

Sninsky and Gelfand, personal communication


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